Nir-Emitting Hemicyanines With Large Stokes' Shifts For Live Cell Imaging: From Lysosome To Mitochondria Selectivity By Substituent Effect

Lysosome imaging without perturbing intracellular activity remains challenging, as the current commercial lysosome probes contain weakly basic amino groups that could perturb lysosome pH. Herein, we illustrate NIR-emitting dyes 2 and 3 (lambda(em) approximate to 700 nm) with very large Stokes' shifts (Delta lambda = 231-246 nm), attributing to the presence of a 2-hydroxyphenyl(benzo[d]oxazol) (HBO) unit that undergoes excited-state intramolecular proton transfer (ESIPT). The structures of 2 and 3 also contain a hemicyanine unit with benzothiazolium and indolium as a terminal group, respectively. Although the fluorescent probe 2 (Phi(fl) approximate to 0.28-0.35 in CH2Cl2) does not contain any basic amino functional group, it exhibits excellent selectivity for staining intracellular lysosomes, showing the potential for long-term in vivo lysosome imaging without "alkalinizing effect." However, probe 3 (Phi(fl) approximate to 0.27, in CH2Cl2) exhibits excellent selectivity toward mitochondria. The observation showed that the terminal group in the hemicyanine unit played an essential role in guiding the intracellular selectivity to different organelles. In addition, the probes also displayed a transparent optical window between 520 and 590 nm, which is useful to achieve multicolor co-staining study, without fluorescence crosstalk that is a common problem on fluorescence microscopes.